生精细胞的体外培养

目的:在体外培养中,观察精原细胞的增殖、分化和形态变化规律。方法:用胶原酶、透明质酸酶和胰蛋白酶分次消化法制取睾丸组织的细胞悬液,密度梯度离心法富集生精细胞,贴壁培养后观察生精细胞的存活、分裂和形态变化,并行AnnexinⅤ-FITC/PI染色,采用激光共聚焦扫描显微镜检测细胞的凋亡情况。结果:睾丸组织细胞悬液中的体细胞在培养中较早贴壁,胞质形成多个突起;精原细胞贴壁较迟,贴壁后呈规则的圆形或卵圆形,以链状或团块状位于体细胞层之上,细胞分裂不完全,相互间以细胞间桥相连。AnnexinⅤ-FITC/PI染色显示,培养第2天时精原细胞已出现凋亡,但数量极少,后逐渐增多。结论:在本实验所提供的体外培养条件下,精原细胞能够存活并发生贴壁、分裂、分化等行为,但同时也有细胞凋亡。

Tumor necrosis factor-alpha induces apoptosis of enterocytes in mice with fulminant hepatic failure

AIM: To explore the alterations of intestinal mucosa morphology, and the effects of tumor necrosis factor a (TNFα) on enterocyte apoptosis in mice with fulminant hepatic failure (FHF). METHODS: Liver damage was induced by lipopolysaccharide (LPS)/TNF-α in D-galactosamine (GaIN) sensitized BALB/c mice. There were 40 mice in normal saline (NS)-treated group, 40 mice in LPS-treated group, 40 mice in GaIN-treated group, 120 mice in GaIN/ LPS-treated group and 120 mice in GaIN/ TNFα-treated group. Each group was divided into five subgroups of eight mice each. Serum samples and liver, intestinal tissues were respectively obtained at 2, 6,9,12 and 24 h after administration. Anti-TNFa monoclonal antibody was injected intravenously into GaIN/LPS-treated mice. Serum TNFα levels were determined by enzyme linked immunosorbent assays (ELISA). Serum ALT levels were determined using an automatic analyzer. The intestinal tissues were studied under light microscope and electron microscope at 2, 6, 9,12 and 24 h in mice with fulminant hepatic failure, respectively. Enterocyte apoptosis was determined by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) method. The expression of tumor necrosis factor receptor 1 (TNFR1) in intestinal tissue was tested by immunohistochemistry Envision Two Steps. RESULTS: Gut mucosa was morphologically normal at all time points in all groups, but typical apoptotic cells could be seen in all experimental groups under electron microscope. Apoptosis rate of gut mucosal epithelial cells were significantly increased at 6, 9 and 12 h, peaked at 12 h in mice with fulminant hepatic failure. TNFa induced apoptosis of enterocytes in mice with FHF. The integrated OD (IOD) levels of TNFa receptor 1 protein expressed in the intestine of mice with GaIN/LPS and GaIN/ TNFα induced FHF at 2, 6, 9, 12 and 24 h after GaIN/LPS and GaIN/TNFα administration were 169.54±52.62/905.79±111.84,11 350.67±2 133.26/28 160.37±4 601.67, 25 781.00±2 277.75/122 352.30±49 412.40, 5 241.53±3 007.24/ 49 157.93±9 804.88, 7 086.13±1 031.15/3 283.45±127.67, respectively, compared with those in control groups (with NS, LPS and GaIN administration, respectively). IOD level of TNFR1 changed significantly at 6, 9 and 12 h after GaIN/LPS and GalN/TNFa administration. The expression of TNFR1 protein was significantly higher at 9 h after GaIN/LPS and GaIN/TNFα administration than that in control groups. Protein expression of TNFR1 was positively correlated with enterocyte apoptosis. CONCLUSION: TNFα can induce apoptosis of enterocytes in mice with FHF. Anti-TNFα IgG can inhibit this role.

Role of cathepsin B-mediated apoptosis in fulminant hepatic failure in mice

AIM: To investigate the pathogenic role of cathepsin B and the protective effect of a cathepsin B inhibitor (CA074Me) in fulminant hepatic failure in mice. METHODS: LPS/D-Gal N was injected into mice of the model group to induce fulminant hepatic failure; the protected group was administered CA-074me for 30 min before LPS/D-Gal N treatment; the normal group was given isochoric physiologic saline. Liver tissue histopathology was determined with HE at 2, 4, 6 and 8 h after Lps/D-Gal injection. Hepatocyte apoptosis was examined by TUNEL method. The expression of cathepsin B in liver tissues was investigated by immunohistochemistry, Western blot and RT-PCR. RESULTS: Compared with the normal group, massive typical hepatocyte apoptosis occurred in the model group; the number of apoptotic cells reached a maximum 6 h after injection. The apoptosis index (AI) in the protected group was clearly reduced (30.4 ± 2.8 vs 18.1 ± 2.0, P < 0.01 ). Cathepsin B activity was markedly increased in drug-treated mice compared with the normal group (P < 0.01). Incubation with LPS/D-Gal N at selected time points resulted in a timedependent increase in cathepsin B activity, and reached a maximum by 8 h. The expression of cathepsin B was significantly decreased in the protected group (P < 0.01). CONCLUSION: Cathepsin B plays an essential role in the pathogenesis of fulminant hepatic failure, and the cathepsin B inhibitor CA-074me can attenuate apoptosis and liver injury.

Glutamine depletion induces murine neonatal melena with increased apoptosis of the intestinal epithelium

AIM:To investigate the possible biological outcome and effect of glutamine depletion in neonatal mice and rodent intestinal epithelial cells.METHODS:We developed three kinds of artificial milk with different amounts of glutamine;Complete amino acid milk (CAM),which is based on maternal mouse milk,glutamine-depleted milk (GDM),and glutaminerich milk (GRM).GRM contains three-fold more glutamine than CAM.Eighty-seven newborn mice were divided into three groups and were fed with either of CAM,GDM,or GRM via a recently improved nipple-bottle system for seven days.After the feeding period,the mice were subjected to macroscopic and microscopic observations by immunohistochemistry for 5-bromo-2'deoxyuridine (BrdU) and Ki-67 as markers of cell proliferation,and for cleaved-caspase-3 as a marker of apoptosis.Moreover,IEC6 rat intestinal epithelial cells were cultured in different concentrations of glutamine and were subject to a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate cell proliferation assay,flow cytometry,and western blotting to examine the biological effect of glutamine on cell growth and apoptosis.RESULTS:During the feeding period,we found colonic hemorrhage in six of 28 GDM-fed mice (21.4%),but not in the GRM-fed mice,with no differences in body weight gain between each group.Microscopic examination showed destruction of microvilli and the disappearance of glycocalyx of the intestinal wall in the colon epithelial tissues taken from GDM-fed mice.Intake of GDM reduced BrdU incorporation (the average percentage of BrdU-positive staining;GRM:13.8%,CAM:10.7%,GDM:1.14%,GRM vs GDM:P < 0.001,CAM vs GDM:P < 0.001) and Ki-67 labeling index (the average percentage of Ki67-positive staining;GRM:24.5%,CAM:22.4% GDM:19.4%,GRM vs GDM:P=0.001,CAM vs GDM:P =0.049),suggesting that glutamine depletion inhibited cell proliferation of intestinal epithelial cells.Glutamine deprivation further caused the deformation of the nuclear membrane and the plasma membrane,accompanied by chromatin degeneration and an absence of fat droplets from the colonic epithelia,indicating that the cells underwent apoptosis.Moreover,immunohistochemical analysis revealed the appearance of cleaved caspase-3 in colonic epithelial cells of GDM-fed mice.Finally,when IEC6 rat intestinal epithelial cells were cultured without glutamine,cell proliferation was significantly suppressed after 24 h (relative cell growth;4 mmol/L:100.0% ± 36.1%,0 mmol/L:25.3% ± 25.0%,P < 0.05),with severe cellular damage.The cells underwent apoptosis,accompanied by increased cell population in sub-G0 phase (4 mmol/L:1.68%,0.4 mmol/L:1.35%,0 mmol/L:5.21%),where dying cells are supposed to accumulate.CONCLUSION:Glutamine is an important alimentary component for the maintenance of intestinal mucosa.Glutamine deprivation can cause instability of the intestinal epithelial alignment by increased apoptosis.

Effect of Angelica keiskei chalcone on the expression of apoptosis-regulating proteins of mice hepatocarcinoma cells

Objective: The aim of our study was to investigate the effect of Angelica keiskei chalcone (AC) on the expression of Caspase-3 and Bax in mice hepatocarcinoma cells. Methods: Fifty mice inoculated hepatocarcinoma 22 cells were divided into five groups, 10 mice per group. Mice were given 5, 20, 40 mg/kg AC daily by mouth in low, middle and high dose groups respectively. Saline were given to the tumor control group by mouth. Twenty mg/kg cytoxan (CTX) by injection every other day were given to the positive control group. Ten days later, all mice were sacrificed. The levels of the Caspase-3 and Bax protein expression were measured by immunohistochemistry method and the proliferation activity of hepatocarcinoma cells was determined by MTT assay. Results: The expression level of Caspase-3 and Bax protein in tumor control group were 5.00% and 4.68%, respectively, and those of the high-dose group were 38.52% and 35.76%. The differences between two groups were significant (P < 0.05). The cell proliferation activity of tumor control group and high-dose group were 1.135 ± 0.032 and 0.716 ± 0.018. The difference was significant (P < 0.05). Conclusion: AC can increase the expression of Caspase-3 and Bax protein, and inhibit the proliferative activity of mice hepatocarcinoma cells.

A critical role of PUMA in maintenance of genomic integrity of murine spermatogonial stem cell precursors after genotoxic stress

Neonatal gonocytes are precursors of spermatogonial stem cells. Preserving their integrity by elimination of damaged germ cells may be crucial to avoid the transmission of genetic alterations to progeny. Using 7-irradiation, we investigated by immunohistochemistry, flow cytometry and real-time PCR components of the death machinery in neonatal gonocytes. Their death was correlated with caspase 3 activation but not with AIF translocation into the nu- cleus. The in vivo contribution of both the extrinsic and the intrinsic pathways was then investigated. We focused on the roles of TRAIL/Death Receptor 5 （DR5） and PUMA. Our results were validated using knockout mice. Whereas DR5 expression was upregulated at the cell surface after radiation, caspase 8 was not activated. However, we detected caspase 9 cleavage associated with cytochrome c release. In mice deficient for PUMA, radiation-induced gonocyte apoptosis was reduced, whereas invalidation of TRAIL had no effect. Overall, our results show that genotoxic stressinduced apoptosis of gonocytes is caspase-dependent and involves almost exclusively the intrinsic pathway. Furthermore, PUMA plays a critical role in the maintenance of genomic integrity of spermatogonial stem cell precursors.

The effect of pre-low-dose X-ray radiation on tumor inhibition of HepG2 cells in tumor-bearing nude mice

Objective:The aim of this study was to discuss the effect of pre-low-dose X-ray radiation on P53,Bcl-2 and apoptosis of HepG2 cells in tumor-bearing nude mouse,and further explore the mechanism of low doses radiation.Methods:HepG2 cells were implanted subcutaneously into nude mice.14 days after the implanting,these mice were divided into 6 groups randomly,S group (sham-irradiation 0 cGy),D1 group (7.5 cGy,dosage rate=7.5 cGy/min),D2 group,(200 cGy,dosage rate=100 cGy/min),D1 + 2 h + D2 group,D1 + 6 h + D2 group and D1 + 12 h + D2 group.Tumor-bearing mice in each experimental group were executed at 24 h after the last irradiation.P53 and Bcl-2 were detected by immunohistochemical staining,the tumor tissues apoptosis were detected in site (Tunel).Results:Each combined exposure groups (D1 + 2 h + D2 group,D1 + 6 h + D2 group and D1 + 12 h + D2 group) compared with the D2 group,the percentages of positive P53 and Bcl-2 were decreased obviously,and the apoptotic indexs were increased (P < 0.01).Conclusion:Pre-low-dose radiation combined with the conventional radiation can increase the apoptosis of tumor tissues by decreasing the expression of P53 and Bcl-2,it can enhance the anti-tumor effect of conventional radiation,and it can have actual clinical significance on supporting radiotherapy.

Effects of Bushenyiqihexue Formula on the Endometrial Gland Apoptosis in Mice with Blastocyst Implantation Dysfunction

Objective: To observe the effects of Bushenyiqihexue Formula (补肾益气和血方 Formula for Tonifying the Kidney, Replenishing qi and Harmonizing Blood, FTKRQHB) on the endometrial gland apoptosis in the mice with blastocyst implantation dysfunction. Methods: The mice with the first-day pregnancy were divided into the control, model and treatment groups, with 30 in each group, and blastocyst implantation dysfunction was induced by subcutaneous injection of mifepristone in the mice of the model and treatment groups. The pregnancy rate and implantation number of blastocysts were measured and the expressions of proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, and activated caspase-3 were detected in all the three groups. Results: The model group had significantly depressed pregnancy rate, implantation number of blastocysts and apoptosis index, and elevated proliferation index of endometrial gland as compared with the control group (P<0.05 or P<0.01). Administration of FTKRQHB (the treatment group) resulted in significant increases in pregnancy rate, implantation number of blastocysts and apoptosis index of the endometrial gland, and a significant decrease in the proliferation index of the endometrial gland as compared with the model group (P<0.05 or P<0.01). The differences in the four indexes between the treatment group and control group were not significant statistically. The Bax and activated caspase-3 expressions in endometrial gland in the model group became significantly lower than that of the control group (P<0.01), whereas those in the treatment group were significant higher than that of the model group (P<0.01). However, the Bax and activated caspase-3 expressions in endometrial gland were similar in both treatment and control groups. Conclusion: Promoting the increases in Bax and activated caspase-3 expressions in the endometrial gland and bringing into balance between apoptosis and proliferation of the glandular cells at the implantation window phase by FTKRQHB may contribute to the effects of promoting the establishment of endometrial receptivity and improving blastocyst implantation dysfunction.

Overexpression of a dominant-negative mutant of SIRT1 in mouse heart causes cardiomyocyte apoptosis and early-onset heart failure

SIRT1,a mammalian ortholog of yeast silent information regulator 2(Sir2),is an NAD+-dependent protein deacetylase that plays a critical role in the regulation of vascular function.The current study aims to investigate the functional significance of deacetylase activity of SIRT1 in heart.Here we show that the early postnatal hearts expressed the highest level of SIRT1deacetylase activity compared to adult and aged hearts.We generated transgenic mice with cardiac-specific expression of a dominant-negative form of the human SIRT1(SIRT1H363Y),which represses endogenous SIRT1 activity.The transgenic mice displayed dilated atrial and ventricular chambers,and died early in the postnatal period.Pathological,echocardiographic and molecular phenotype confirmed the presence of dilated cardiomyopathy.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis revealed a greater abundance of apoptotic nuclei in the hearts of transgenic mice.Furthermore,we show that cardiomyocyte apoptosis caused by suppression of SIRT1 activity is,at least in part,due to increased p53acetylation and upregulated Bax expression.These results indicate that dominant negative form of SIRT1(SIRT1H363Y)overexpression in mouse hearts causes cardiomyocyte apoptosis and early-onset heart failure,suggesting a critical role of SIRT1 in preserving normal cardiac development during the early postnatal period.

Effects of Electroacupuncture on Hippocampal and Cortical Apoptosis in A Mouse Model of Cerebral Ischemia-reperfusion Injury

Objective: To observe the effects of electroacupuncture on hippocampal and cortical apoptosis in a mouse model of cerebral ischemia-reperfusion injury. Methods: Mouse models established by repeated cerebral ischemia-reperfusion, followed by electroacupuncture at Shenshu, Geshu, and Baihui points. The control group mice were intragastrically administered Hydergine. On day 1 and 7 post-treatment, hippocampal and cortical apoptosis was detected by terminal-deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL), and apoptosis images in the hippocampal CA1 zone and cortical area were analyzed. Results: In the model group, apoptotic cells were detected one day after treatment and some cellular fibers were disarrayed. By day 7 post-treatment, there was an increase in the number of apoptotic cells in the hippocampal CA1 region. In addition, there were apoptotic cells in the cortical area, the cortical layers were thinner with localized neuronal loss and sieve-like lymphocyte infiltration, as well as glial cell proliferation and visible infarct lesions. However, in the Hydergine and electroacupuncture groups, there was a small number of apoptotic cells. At 7 days post-treatment in the model group, field number, numerical density on area, and surface density were increased. However, in the Hydergine and electroacupuncture groups these parameters were decreased (P<0.01), with a significant difference between the two treatment groups (P<0.01). Conclusion: Electroacupuncture treatment inhibited apoptosis and provided neuroprotection.

Inhibitive effect on apoptosis in splenic lymphocytes of mice pretreated with Lingzhi (GanodermaLucidum) spores

OBJECTIVE： To investigate how the pretreatment of mice with Ganoderma spores affected the apop- tosis of their splenic lymphocytes induced by dexa- methasone after 19 days treatment. METHODS： Sixty Kunming mice were randomly di- vided into six groups： blank control groupdrenched with normal saline; a drug control group drenched with 150 mg/mL Ganoderma spores; a model group treated with saline; a low dose group with S0 mg/mL Ganoderma spores; a moderate dose group with 100 mg/mL Ganoderma spores; and a high dose group with 150 mg/mL Ganoderma spores. The effect of Ganoderma spores on apoptosis in spleen lymphocytes was analyzed. All groups were treated for 19 days. On day 20, the model group and the 3 treatment groups were intraperitoneally injected dexamethasone to induce apoptosis. Splenic index and apoptosis indes were employed to measure cell apoptosis RESULTS： The results showed that Ganoderma spores reduced the splenic index to different de- grees in each group and the best effect was seen in the high dose group （P〈0.05）. Terminal dexynucleo- tidyl transferase （TdT）-mediated 2＇-Deoxyuridine 5＇-Triphosphate nick end labeling staining revealed that the apoptotic index in all groups administered Ganoderma spores differed significantly from the model group, and a dose-response was observed. Flow cytometric analysis indicated that spleen lym- phocyte apoptosis in the model group was exten- sive. Each dose of Ganoderma spores inhibited dexamethasone-induced apoptosis in spleen lym- phocytes, and a dose-response was observed as well. The highest dose of Ganoderma spores de- creased Malondialdehyde content in serum in- duced by dexamethasone （P〈0.05）. CONCLUSION： The findings imply that the pretreat- ment of the mice with Ganoderma spores could re- duce the apoptosis rate induced by dexametha- sone in their splenic lymphocytes.

Effect of quercetin on the expression of Bcl-2/Bax apoptotic proteins in endometrial cells of lipopolysaccharide-induced-abortion mice

OBJECTIVE: To explore the effect of quercetin on the expressions of Bcl-2/Bax apoptotic proteins in endometrial cells in mice with abortion induced by lipopolysaccharide.METHODS: For in vivo experiment, twenty five Kunming mice were randomly divided into five groups at day 4 of pregnancy, with 5 mice per group. The mice were treated with lipopolysaccharide(LPS)through tail vein intravenous injection at day 4 of pregnancy, followed by different concentrations of quercetin by oral gavage consecutively at days 5 to6 of pregnancy. On day 7 of gestation, the mice were sacrificed and the histopathological changes of the uterus tissues were observed. Immunohistochemical staining was applied to the detection of Bcl-2/Bax apoptotic proteins in the endometrial cells. For in vitro experiment, the primary endometrial cells werecultured using a uterus tissue mass culturing method sampled at day 4.5 of pregnancy. The cells were treated with LPS with or without different dosages of quercetin, respectively, for 12 h after 80% confluence. The expression of Bcl-2/Bax apoptotic proteins were detected by western blotting.RESULTS: Both the in vivo and in vitro experiments showed decreased expression of Bcl-2 and enhanced expression of Bax after LPS treatment, leading to a decreased Bcl-2/Bax ratio. The expression of Bcl-2 significantly increased while the expression of Bax was significantly elevated, in the LPS plus quercetin group compared to the LPS only group.CONCLUSION: These results suggest that quercetin has protective effect by partially regulating the expression of Bcl-2/Bax proteins, which in turn inhibits endometrial cell apoptosis and benefits the embryo implantation.

Apoptosis within mouse eye induced by Toxoplasma gondii

Objective To investigate apoptosis induced by Toxoplasma gondii (T. gondii) in eyes of C57BL/6 (B6) mice. Methods Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) technique and pathological changes within eyes were analyzed at different time points after intraocular inoculation of either 50 or 500 of tachyzoites. Results In eyes that received 50 tachyzoites, a few apoptotic inflammatory cells in the anterior chamber and keratocytes in the cornea were seen at days 1 and 2, but no apoptosis was detected 4 days after inoculation. Significantly greater apoptosis of inflammatory cells was observed in the anterior chamber and in the vitreous of eyes injected with 500 parasites. Apoptosis of inflammatory cells in the anterior chamber and of keratocytes in the cornea was seen at day 1. The apoptotic stromal keratocytes strikingly increased at day 4. There were a number of apoptotic inflammatory cells in the vitreous at day 2, and a few apoptotic retinal cells along the internal limiting membrane and the nerve fiber layer of the retina 4 days after inoculation. Conclusion These results suggest that apoptosis of inflammatory cells infiltrated eye infected with this parasite may be a mechanism of eliminating the organism.